Protein reductive methylation

RA Houghten, CH Li. Reduction of sulfoxides in peptides and proteins. (/V-methyl-sulfamylacetamide) Anal Biochem 98, 36, 1979. 

Naranmandura, H Suzuki, N. and Suzuki, K.T. (2006) Trivalent arsenicals are bound to proteins during reductive methylation. Chemical Research in Toxicology, 19(8), 1010-18. 

Physical or chemical modification of a substrate may additionally selectively affect transformation or uptake Keil and Kirchman (1992) compared the degradation of Rubisco uniformly labeled with 3H amino acids produced via in vitro translation to Rubisco that was reductively methylated with 3H-methane. Although both Rubisco preparations were hydrolyzed to lower molecular weights at approximately the same rate, little of the methylated protein was assimilated or respired. The presence of one substrate may also inhibit uptake of another, as has been demonstrated for anaerobic rumen bacteria. Transport and metabolism of the monosaccharides xylose and arabinose were strongly reduced in Ruminococcus albus in the presence of cellobiose (a disaccharide of glucose), likely because of repression of pentose utilization in the presence of the disaccharide. Glucose, in contrast, competitively inhibited xylose transport and showed noncompetitive inhibition of arabinose transport, likely because of inactivation of arabinose permease (Thurston et al., 1994). 

The effect of protein methylation (20% modification) on the stability of casein micelles was evaluated. The stability of the clJk micelle system towards Ca2+ precipitation was decreased when either or both proteins were methylated (see Figure 1). Methylation also resulted in a slight reduction in the stability of the /3-k casein micelle system (see Figure 2). 

The temperature-sensitive precipitation of unmodified and methylated /3-caseins in the presence of calcium was measured also (see Figure 3). Methylation caused an increase of up to 3°C in the precipitation temperature of calcium /3-caseinate. Results from rennet clotting of an asi-K casein micelle system indicated that replacing native asi-casein with the reductively methylated protein had little influence on clotting time, while replacing K-casein with its reductively methylated derivative re-... 

We chose to prepare 14C-methyl-K-casein (M-k-C) as a tracer because of the important role of K-casein in stabilizing casein micelles (8) and because K-casein is known to participate in heat-induced interactions with whey proteins, thereby influencing the heat stability of milk (9). The reductive methylation radiolabeling procedure used low concentrations of reagents (10) and resulted in M-k-C containing approximately 1 fiinol of 14C-methyl groups for every micromole of protein monomer (about 3 /xCi/mg). When tracer M-k-C was added to skim milk, and trichloroacetic acid was added to a concentration of 2%, about 1% of the radioactivity remained soluble. After clotting of the milk with excess... 

C-Methyl-/3-Casein. 14C-Labeled proteins prepared by reductive methylation have potential as substrates in the study of proteolytic enzymes. A serious limitation is that complete methylation of lysine residues results in inhibition of proteolysis by enzymes with trypsin-like specificity (13). It was interesting to determine whether this problem could be overcome by incomplete methylation which left unaltered most of the lysine residues in more or less random distribution throughout the protein. /3-Casein was selected as a suitable protein for this study since it is cleaved by trypsin-like enzymes to well characterized fragments, the y-caseins, in addition to less well characterized fragments, the proteose peptones. We anticipated that this type of study could provide a basis for a general investigation of milk protein transformation by the native milk proteinase which has a specificity similar to trypsin (14). 

Another tritium labeling method is the reductive methylation 128,129) dehyde is allowed to combine with amino groups of a protein and is then reduced using sodium borohydride. The details will be given later in a paragraph concerned with labeling. 

Foreign labeling in vitro is certainly very widely used. The most important labeling methods which have been applied for labeling almost all important proteins are halogenation (iodination or bromation) and conjugation (reductive methylation) alkylation and acylation 3 )... 

In either case, a reservoir of a solution with a higher concentration of precipitant is present in the same compartment. Water evaporates from the samples into the larger reservoir, concentrating the protein and causing its crystallization. Crystals grow slightly better in a spacecraft than on Earth. ° ° Some proteins, notably myosin from muscle, crystallize well only after reductive methylation of all lysine side chains to dimethyllysine with formaldehyde and sodium borohydride (Eq. 3-34). ... 

Posttranscriptional processing of tRNA requires several distinct steps, as summarized in Figure 25.8. First, the 50 and 30 ends must be cleaved to release the tRNA sequence from the larger precursor transcript and introns must be removed if they are present. Second, the required CCA charging sequence at the 30 end of tRNA must sometimes be added by a nucleotidyl transferase. Third, all tRNAs contain a large number of modified bases which result from reductions, methylations, and deaminations. These modifications can affect codon recognition by the tRNAs during protein synthesis (Chapter 26). 

Thus, modification of lysine residues with imido esters (150) by reductive methylation (158,157) or with other reagents (154) is a useful way of introducing radioactive labels into the protein without destroying the enzymic activity. 

UMP, the product of both the biosynthetic and salvage pathways, serves as the precursor for all other piyrimidine nudeotides. It is converted to UDP and UTP by nudeotide kinases, UTP is aminated to CTP by CTP s)mthetase (CTPS), deoxyribonudeotides are synthesized fix>m ribonudeoside diphosphates by ribonudeotide reductase (RIO, and thymidylate nudeotides ate produced via reductive methylation of dUMP utilizing N, N °-meth)denetetrahydtofolate as the methyl donor, a reaaion catalyzed by a bifuncdonal dihydrofolate teductase-thymidylate synthase (DHFR-TS) protein. 

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